Colorimeter

• It is a most common analytical technique used

in biochemical estimation in clinical laboratory

• It involves the quantitative estimation of colour

• A substance to be estimated colorimetrically

must b e colored or colored complexes through
the addition of reagents
• Colored substance absorb light in relation to
their color intensity
• The colour intensity will be proportional to the
concentration of colored substance
Principle
Colored solution have the property of absorbing
certain wavelength of light when a monochromatic
light is passed through them

The amount of light absorbed or transmitted by a

colored solution is based on two laws
• Beer's law
• Lambert’s law
• Beer's law:
The amount of light absorbed by a colored
solution is directly proportional to the
concentration of substance In the colored solution

• Lambert's law:
The amount of light absorbed by a colored
solution depends on path length or thickness of the
colored solution
Combined Beer's law & Lambert's law
Amount of light transmitted through a colored
solution decreases exponentially with increase in
concentration of colored solution and decrease in
the path length of thickness of the colored
solution
 Instrumentation
• Light source
• Slit
• Lens
• Filter
• Sample holder (cuvette)
• Detector (photocell)
• Output
• Light source : In colorimeter source of visible
light is a tungsten filament lamp wavelength
ranges from (400-700 nm)

• Slit : it is adjustable which allows only beam of

light to pass through it .it prevents unwanted
light

• Condensing lens: light after passing through

slit falls on condenser lense which gives a
parallel beam of light
• Filter : filter are used for selecting light of narrow
wavelength it allow only monochromatic light to
pass through it

• Sample holder (cuvette) : the monochromatic

light from the filter passes through the colored
solution placed in a cuvette

• The coloured solution in the cuvette absorbs part

of light & remaining is allow to fall on the
detector
• Detector (photo cell) : Detector are photosensitive
elements which converts light energy in to electrical
energy

• The electrical signal generated is directly

proportional to intensity of light falling on the
detector

• Output : the electrical signal generated in photocell

is measured by galvanometer, which displays
percent transmission &optical density
Spectophotometer
• It is a measurement that measures the amount of light
absorbed by a sample after it is passed trough its
solution
• Principle
• Each compound absorbs or transmits light over a
certain range of wavelength

• The light absorption is directly proportional to the

concentration of compound in the sample

• As concentration increases, light absorption increases

linearly but light transmission decreases exponentially
• Based on wavelength of light used it can be
classified in to threee types
• Visible spectrometer:
Use visible range (400-700nm) of
electromagnetic radiation spectrum
• UV spectrometer:
Use UV range light over (180-400nm)
• IR spectrometer
Use light IR range (700-800) electromagnetic
spectrum
• It consists of two devices
• Spectrometer: A device that produce,typically
dispence and measure the light

• Photometer : it ondicates the photo electric

detector measure the light
 Instruments
• Light source
• Monochromoter (it consists of)
• Entrance Slit
• Collimating Lens
• Dispersion elemens
• Prism
• Exit slit
• Sample holder (cuvette)
• Detector (photocell)
• Output
• Light source: to provide a sufficient of light to
be passed through the sample

• Tungsten lamp: It is most common light

source
wavelength range from 330 to900nm long life
about 12000hr

• Hydrogen lamp: For UV region this lamps are

frequently used range from 200 to 450nm more
stable has long life about 500hr
• Xenon lamp: It emit both UV –visible
wavelength

• The wavelength range from 190nm-1000nm

• Long life
Dispersion devices:

• Monochromator: Accepts polychromatic input

light from a lamp and out puts monochromatic
light
• it is 3 parts
1.Entrance slit
2.prism
3.Exit slit
• Cuvette: Cuvettes are made from plastic, glass,
or quartz.
• It should be clear without impurities that
might affect spectroscopic reading.
• Do not allow samples to sit in a cuvette for a
long period of time.
• Wash cuvettes immediately after use.
• Detector : Convert radient energy to electrical
energy generate signal is directly proportional
to intensity of light

• Output : the data from a detector are

displayed by a read out device, the output can
also be transmitted to a computer or printer
 Applications
• Determines the presence and concentrations
of samples.
• Determines the purity of a sample.
• Look at the change of samples over time.